Twiner, M, J, University of Western Ontario, London, Canada, mtwiner@uwo.ca

Trick, C, G, University of Western Ontario, London, Canada, trick@uwo.ca

THE DETECTION OF NITRIC OXIDE (NO) PRODUCTION BY HETEROSIGMA AKASHIWO USING THE FLUORESCENT DYE DAF-FM DIACETATE AND FLOW CYTOMETRY

Increasing occurrences of bloom outbreaks of the marine flagellate Heterosigma akashiwo have been reported over the past few decades along coastal regions around the world, causing devastating effects both environmentally and economically. Due to the bloom and bust nature of H. akashiwo, it's high levels of production of reactive oxygen species, and its high density of chloroplasts, Heterosigma is likely to produce considerable amounts of the greenhouse gas, NO. Our objective was to monitor the physiological production of NO by growing and senescent H. akashiwo cells in different environmental conditions.
NO was detected in individual H. akashiwo cells using a fluorescent dye indicator coupled with flow cytometry analysis. DAF-FM diacetate (4-amino-5-methylamino-2`7`-difluorofluorescein) solution in DMSO efficiently measures NO at low concentrations (Kojima et al Anal Chem 70, 2446-2453 1998 ). Experiments investigating the production of NO by H. akashiwo were conducted by altering environmental conditions, such as light intensity and nutrient conditions (N and Fe supply). Growth and NO production were monitored in conditions with differing N:P ratios and Fe availability using the artificial chelator DFB. Our experiments have suggested a substantial increase in NO production from Fe-limited cells.